共表达猪瘟病毒E2基因和猪白细胞介素2基因的重组腺病毒的构建及其免疫原性研究

Construction and Immunogenicity of a Recombinant Adenovirus Co-expressing the E2 Protein of Classical Swine Fever Virus and the Porcine Interleukin 2 in Rabbits

为研制猪瘟活载体疫苗,构建共表达猪瘟病毒E2基因和猪白细胞介素2基因的重组腺病毒并研究其免疫原性。应用PCR方法从pMD19-T-E2质粒和pMD19-T-pIL2质粒中分别扩增E2基因和pIL-2基因,将扩增的全长E2基因和pIL-2的编码区基因序列通过柔性接头序列(5个甘氨酸密码子)串联,插入腺病毒穿梭载体AdTrack中,在受体菌中与骨架载体AdEasy同源重组。重组质粒AdEasy-E2-pIL-2转染HEK293细胞,包装出重组腺病毒rAd-E2-pIL-2。用rAd-E2-pIL-2免疫家兔,经兔体交互免疫试验评定其免疫效果。结果显示,经RT-PCR和West-ern blot检测,成功构建了rAd-E2-pIL-2,重组病毒滴度达108.12PFU/mL。rAd-E2-pIL-2接种家兔后刺激接种兔产生猪瘟病毒特异性抗体,淋巴细胞转化试验结果显示猪瘟病毒诱导兔淋巴细胞特异性增殖,攻毒后rAd-E2-pIL-2接种兔和猪瘟病毒C株接种兔均未出现定型热反应。研究结果表明,rAd-E2-pIL-2免疫兔可以预防猪瘟病毒C株接种引发的体温反应,rAd-E2-pIL-2可望成为猪瘟候选疫苗。

To construct a recombinant adenovirus co-expressing the E2 protein of classical swine fever virus（CSFV） and the porcine interleukin 2（pIL-2）,the CSFV E2 gene and pIL-2 gene were amplified respectively from the plasmids pMD19-T-E2 and pMD19-T-pIL-2 by PCR.E2-pIL-2 fusion gene was obtained by using 5 consecutive glycine codons as a linker and cloned into the adenoviral shuttle plasmid AdTrack.The AdTrack-E2-pIL-2 was linearized and transformed into E.coli BJ5183 with the backbone plasmid AdEasy-1.The resultant recombinant plasmid AdEasy-E2-pIL-2 was transfected into the 293 cells where the recombinant adenovirus rAd-E2-pIL-2 was produced.The immunogenicity of rAd-E2-pIL-2 was evaluated in rabbits.The results of RT-PCR and Western-blotting showed that rAd-E2-pIL-2 could carry and express E2 and pIL-2 proteins.The titer of the rAd-E2-pIL-2 was 108.12 PFU/mL.After immunized with rAd-E2-pIL-2,The injected rabbits developed a high level of CSFV specific antibodies.Regular fever was not detected in the rAd-E2-pIL-2-immunized rabbits upon challenge with CSFV C stain,and specific lymphoproliferative responses to the CSFV was detected in the lymphocytes from the immunized rabbits.In conclusion,rAd-E2-pIL-2 was constructed successfully and it could be an attractive vaccine candidate against CSFV.